Physical and chemical factors influencing the germination of Clostridium difficile spores

AIMS: To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. METHODS AND RESULTS: Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. CONCLUSIONS: This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.

Antimicrobial efficacy of copper surfaces against spores and vegetative cells of Clostridium difficile: the germination theory

OBJECTIVES: Persistent contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of C. difficile-associated diarrhoea (CDAD) in the clinical setting. In recent years, the antimicrobial efficacy of metal surfaces has been investigated against microorganisms including methicillin-resistant Staphylococcus aureus. This study compared the survival of C. difficile on stainless steel, a metal contact surface widely used in hospitals, and copper surfaces. METHODS: Antimicrobial efficacy was assessed using a carrier test method against dormant spores, germinating spores and vegetative cells of C. difficile (NCTC 11204 and ribotype 027) over a 3 h period in the presence and absence of organic matter. RESULTS: Copper metal eliminated all vegetative cells of C. difficile within 30 min, compared with stainless steel which demonstrated no antimicrobial activity (P < 0.05). Copper significantly reduced the viability of spores of C. difficile exposed to the germinant (sodium taurocholate) in aerobic conditions within 60 min (P < 0.05) while achieving a >or=2.5 log reduction (99.8% reduction) at 3 h. Organic material did not reduce the antimicrobial efficacy of the copper surface (P > 0.05).

Histidine acts as a co-germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co-germinant. The aim of this study was to determine whether any other amino acids act as co-germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re-germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co-germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l-1. Conclusions: Histidine was found to be a co-germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.

Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.

The development of filamentous fungi in submerged culture

During the 24 hour period following inoculation, aggregation of spores and sporelings can have an important effect on the subsequent growth of filamentous fungi in submerged culture. This early phase of growth does not appear to have received much attention, and it was for this reason that the author's research was started. The aggregation, germination and early growth of the filamentous fungus Aspergillus niger have been followed in aerated tower fermenters, by microscopic examination. By studying many individual sporelings it has been possible to estimate the specific growth rate and germination times, and then to assess the branching characteristics of the fungus over a period of from 1 to 10 hours after germination. The results have been incorporated into computer models to simulate the development of the physical structure of individual and aggregated sporelings. Following germination, and an initial rapid growth phase, fungi were found to grow exponentially: in the case of A.niger the mean germination time was about 5 hours and the doubling time was as short as 1.5 hours. Branching also followed an exponential pattern and appeared to be related to hyphal length. Using a simple hypothesis for growth along with empirical parameters, typical fungal structures were generated using the computer models : these compared well with actual sporelings observed under the microscope. Preliminary work suggested that the techniques used in this research could be successfully applied to a range of filamentous fungi.

Sporicidal activity of two disinfectants against Clostridium difficile spores

The sporicidal activity of an odour-free peracetic acid-based disinfectant (Wofasteril®) and a widely-used dichloroisocyanurate preparation (Chlor-clean®) was assessed against spores of the hyper-virulent strain of Clostridium difficile (ribotype 027), in the presence and absence of organic matter. In environmentally clean conditions, dichloroisocyanurate achieved a >3 log10 reduction in 3 minutes, but a minimum contact time of 9 minutes was required to reduce the viable spore load to below detection levels. Peracetic acid achieved a >3 log10 reduction in 30 minutes and was overall significantly less effective (P<0.05). However, in the presence of organic matter - which reflects the true clinical environment - there was no significant difference between the sporicidal activity of dichloroisocyanurate and peracetic acid over a 60-minute period (P=0.188). Given the greater occupational health hazards generally associated with chlorine-releasing agents, odour-free peracetic acid-based disinfectants may offer a suitable alternative for environmental disinfection.

Growth, sporulation and spore properties of bacillus stearothemophilus produced in chemically defined media

The nutritional requirements for the vegetative growth of B. stearothermophilus strains NCIB 8919, NCTC lO,OO3 (wild) were found to be DL-methionine, biotin, nicotinic acid, thiamin, glucose and mineral salts. Strains NCIB 8920 required in addition L-tryptophan. B. stearothermophilus NCTC lO,OO3 (mutant) grew in a medium containing only glucose and mineral salts. Separate chemically defined media for the growth of Bacillus stearothermophilus strains NCIB 8919, 8920, NCTC lO,OO3 (wild) and NCTC lO,OO3 (mutant) were developed. Optimally aerated culture of B. stearothermonhilus NCTC lO,OO3(mutant) required 1.0 x 10-4 M. Mn2+ and 2.4 x 10-3 M. glutamic acid for optimal sporulation. Specific nutrient depletion of growth affected percentage sporulation. Spore suspensions of B. stearothermophilus NCTC 10,003 (mutant) were prepared from media in which sulphate (SO4-), nitrogen (N-),phosphate (Po4-), carbon (C-), magnesium-carbon simultaneously (Ng-C-) depleted growth. The heat resistance, dormancy and chemistry of these spores varied considerably. B. stearothermophilus NCTC 10,003 10,00310,00(mutant) spores prepared from carbon depleted cultures containing high and low concentrations of calcium, iron or manganese showed variations in heat resistance,dormancy and chemical composition. Progressive increase in the concentration of medium calciumfrom 1.0 X 10-5  M to 1.4 X 10-4 M. progressively increased theheat resistance of B. stearothermophilus NCTC 10,003 (mutant) spores prepared from nitrogen depleted cultures (N-). The thermodynamic functions for germination rate, magnesium and manganese release of N- and SO4- spores were within the range expected of enzymic reactions. The thermodynamic functions for the breaking of dormancy in SO4- spores and that for the release of D.P.A. were identical. Sublethal heating of SO4- spores (96.5°C and below) induced dormancy in these spores, whereas heating above 96.5°C gave rise to heat activation. Pooled results of the chemical analyses of all spore types studied showed that the concentration of D.P.A. and calcium were positively related to heat resistance whereas magnesium concentration and Mg/Ca molar ratio were inversely proportional to heat resistance.